Name: Forfatter Spotlight: Udforskning af varmechokproteiner i malaria- og tuberkuloseinfektioner
Uploaded: 2024-03-08T14:25:00
Description: Watch this Scientific Journal Video about Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections at JoVE.com

Arbejdet blev støttet med tilskudsmidler fra International Centre for Genetic Engineering and Biotechnology (ICGEB) bevillingsnummer, HDI / CRP / 012, Forskningsdirektoratet ved University of Venda, bevilling I595, Institut for Videnskab og Innovation (DSI) og National Research Foundation (NRF) i Sydafrika (bevillingsnumre, 75464 &; 92598) tildelt AS.

1. Forvandling
BEMÆRK: Brug sterilt glas til dyrkning, pipettespidser og frisklavede og autoklaverede medier. Forbered kulturer af E. coli-cellerne i 2x gærtrypton (YT) [1,6% trypton (w / v), 1% gærekstrakt (w / v), 0,5% NaCl (w / v), 1,5% agar (w / v)] agar. Generelle reagenser, der anvendes i protokollen og deres kilder, findes i materialetabellen.
<ol><li>Mærk 2,0 ml mikrocentrifugeglas og alikvote 50 μL kompetente E. coli dnaK756-celler...

Vurdering af Hsp70 Chaperon-aktivitet ved hjælp af Escherichia coli dnaK756-celler

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	<li>Bukau, B., Deuerling, E., Pfund, C., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+newly+synthesized+proteins+into+shape.">Getting newly synthesized proteins into shape.</a> Cell. 101 (2), 119-122 (2000).</li><li>Shonhai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodial+heat+shock+proteins:+targets+for+chemotherapy.">Plasmodial heat shock proteins: targets for chemotherapy.</a> FEMS Microbiol. Immunol. 58 (1), 61-74 (2010).</li><li>Mogk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+thermolabile+Escherichia+coli+proteins:+prevention+and+reversion+of+aggregation+by+DnaK+and+ClpB.">Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB.</a> EMBO J. 18 (24), 6934-6949 (1999).</li><li>Edkins, A. L., Boshoff, A., Shonhai, A., Picard, D., Blatch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+structural+and+functional+features+of+molecular+chaperones.+Heat+shock+proteins+of+malaria.">General structural and functional features of molecular chaperones. Heat shock proteins of malaria.</a> Adv Exp Med Biol. , (2021).</li><li>Bertelsen, E. B., Chang, L., Gestwicki, J. E., Zuiderweg, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+conformation+of+wild-type+E.+coli.+Hsp70+(DnaK)+chaperone+complexed+with+ADP+and+substrate.">Solution conformation of wild-type E. coli. Hsp70 (DnaK) chaperone complexed with ADP and substrate.</a> PNAS. 106 (21), 8471-8476 (2009).</li><li>Shonhai, A., Boshoff, A., Blatch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+heat+shock+protein+70+is+able+to+suppress+the+thermosensitivity+of+an+Escherichia+coli+DnaK+mutant+strain.">Plasmodium falciparum heat shock protein 70 is able to suppress the thermosensitivity of an Escherichia coli DnaK mutant strain.</a> Mol Genet Genomics. 274, 70-78 (2005).</li><li>Shonhai, A., Botha, M., de Beer, T. A., Boshoff, A., Blatch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+study+of+a+Plasmodium+falciparum+Hsp70+using+three-dimensional+modelling+and+in+vitro+analyses.">Structure-function study of a Plasmodium falciparum Hsp70 using three-dimensional modelling and in vitro analyses.</a> Protein Pept Lett. 15 (10), 1117-1125 (2008).</li><li>Cockburn, I. L., Boshoff, A., Pesce, E. -. R., Blatch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+modulation+of+plasmodial+Hsp70s+by+small+molecules+with+antimalarial+activity.">Selective modulation of plasmodial Hsp70s by small molecules with antimalarial activity.</a> Biol Chem. 395 (11), 1353-1362 (2014).</li><li>Makhoba, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+chimeric+Hsp70+to+enhance+the+quality+of+recombinant+Plasmodium+falciparum+s-adenosylmethionine+decarboxylase+protein+produced+in+Escherichia+coli.">Use of a chimeric Hsp70 to enhance the quality of recombinant Plasmodium falciparum s-adenosylmethionine decarboxylase protein produced in Escherichia coli.</a> PLoS One. 11 (3), 0152626 (2016).</li><li>Bukau, B., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+defects+caused+by+deletion+of+the+Escherichia+coli+dnaK+gene+indicate+roles+for+heat+shock+protein+in+normal+metabolism.">Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism.</a> J Bact. 171 (5), 2337-2346 (1989).</li><li>Makumire, S., Revaprasadu, N., Shonhai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DnaK+protein+alleviates+toxicity+induced+by+citrate-coated+gold+nanoparticles+in+Escherichia+coli.">DnaK protein alleviates toxicity induced by citrate-coated gold nanoparticles in Escherichia coli.</a> PLoS One. 10 (4), 0121243 (2015).</li><li>Spence, J., Cegielska, A., Georgopoulos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Escherichia+coli+heat+shock+proteins+DnaK+and+HtpG+(C62.+5)+in+response+to+nutritional+deprivation.">Role of Escherichia coli heat shock proteins DnaK and HtpG (C62. 5) in response to nutritional deprivation.</a> J Bact. 172 (12), 7157-7166 (1990).</li><li>Mayer, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistep+mechanism+of+substrate+binding+determines+chaperone+activity+of+Hsp70.">Multistep mechanism of substrate binding determines chaperone activity of Hsp70.</a> Nat Struct Biol. 7 (7), 586-593 (2000).</li><li>Georgopoulos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+bacterial+gene+(groP+C)+which+affects+&#955;+DNA+replication.">A new bacterial gene (groP C) which affects &#955; DNA replication.</a> Mol Genet Genomics. 151 (1), 35-39 (1977).</li><li>Tilly, K., McKittrick, N., Zylicz, M., Georgopoulos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dnaK+protein+modulates+the+heat-shock+response+of+Escherichia+coli.">The dnaK protein modulates the heat-shock response of Escherichia coli.</a> Cell. 34 (2), 641-646 (1983).</li><li>Buchberger, A., Gassler, C. S., Buttner, M., McMacken, R., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+defects+of+the+DnaK756+mutant+chaperone+of+Escherichia+coli+indicate+distinct+roles+for+amino-and+carboxyl-terminal+residues+in+substrate+and+co-chaperone+interaction+and+interdomain+communication.">Functional defects of the DnaK756 mutant chaperone of Escherichia coli indicate distinct roles for amino-and carboxyl-terminal residues in substrate and co-chaperone interaction and interdomain communication.</a> J Biol Chem. 274 (53), 38017-38026 (1999).</li><li>Taj, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+as+a+model+organism.">Escherichia coli as a model organism.</a> Int J Eng Res. 3 (2), 1-8 (2014).</li><li>Sato, S., Wilson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle-specific+cochaperonins+in+apicomplexan+parasites.">Organelle-specific cochaperonins in apicomplexan parasites.</a> Mol Biochem Parasitol. 141 (2), 133-143 (2005).</li><li>Molecular characterisation of the chaperone properties of Plasmodium falciparum. heat shock protein 70. Rhodes University Available from: <a target="_blank" href="https://commons.ru.ac.za/vital/access/manager/Repository/vital:3977?site_name=Rhodes+University">https://commons.ru.ac.za/vital/access/manager/Repository/vital:3977?site_name=Rhodes+University</a> (2007)</li><li>Makumire, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+GGMP+repeat+segments+of+Plasmodium+falciparum+Hsp70-1+compromises+chaperone+function+and+Hop+co-chaperone+binding.">Mutation of GGMP repeat segments of Plasmodium falciparum Hsp70-1 compromises chaperone function and Hop co-chaperone binding.</a> Int J Mol Sci. 22 (4), 2226 (2021).</li><li>Nitika, P. C. M., Truman, A. W., Truttmann, M. 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Protokollen demonstrerer nytten af E. coli dnaK756-cellertil at udforske chaperonfunktionen af heterologt udtrykt Hsp70. Dette assay kunne anvendes til screening af hæmmere rettet mod Hsp70-funktionen i cellulo. En begrænsning ved denne metode er imidlertid, at Hsp70'er, der ikke er i stand til at erstatte DnaK i E. coli , ikke er kompatible med dette assay. Manglende posttranslationel modifikation21 af nogle ikke-indfødte Hsp70'er kan forklare deres manglende funktio...

Varmechokproteiner spiller en vigtig rolle som molekylære chaperoner ved at lette proteinfoldning, forhindre proteinaggregering og vende proteinfejlfoldning 1,2. Varmechokprotein 70 (Hsp70) er en af de mest fremtrædende molekylære chaperoner, der spiller en central rolle i proteinhomeostase 3,4. DnaK er E. coli Hsp70 homolog5.
Forskellige biofysiske, biokemiske og cellebaserede assays er blevet udviklet til at udforske chaperonaktiviteten af Hsp70 og til at screene for inhibitorer rettet mod denne chaperon 6,7,8. Hsp70 er et meget konserveret protein. Af denne grund er flere Hsp70'er af eukaryote organismer, såsom Plasmodium falciparum (malariaens hovedmiddel), rapporteret at erstatte DnaK-funktionen i E. coli 6,9. På denne måde er der udviklet et E. coli-baseret komplementationsassay, der involverer den heterologe ekspression af Hsp70'er i E. coli for at udforske deres cytoprotektive funktion. Typisk involverer dette assay udnyttelsen af E. coli-celler, der enten mangler DnaK, eller som udtrykker en indfødt DnaK, der er funktionelt kompromitteret. Mens DnaK ikke er afgørende for E. coli-vækst under normale forhold, bliver det vigtigt, når cellerne dyrkes under stressende forhold som forhøjede temperaturer eller andre former for stress10,11.
E. coli-stammer, der er udviklet til at studere Hsp70-funktion ved hjælp af et komplementationsassay, omfatter E. coli dnaK103 (BB2393 [C600 dnaK103 (Am) thr:: Tn10]) og E. coli dnaK756. E. coli dnaK103-celler producerer en afkortet DnaK, der ikke er funktionel, og som sådan vokser cellerne tilstrækkeligt ved 30 °C, mens stammen er følsom over for kulde og varmestress 12,13. Tilsvarende vokser E. coli dnaK756/BB2362-stammen (dnaK756 recA::TcR Pdm1,1) ikke over 40 °C14,15. E. coli dnaK756-stammen udtrykker en mutant native DnaK (DnaK756) kendetegnet ved tre glycin-til-aspartatsubstitutioner i positionerne 32, 455 og 468, hvilket giver anledning til kompromitterede proteostatiske resultater. Følgelig er denne stamme resistent over for bakteriofag λ DNA14. Derudover udviser E. coli dnaK756 forhøjet ATPase-aktivitet, mens dens affinitet for nukleotidbytterfaktoren, GrpE, reduceres16. E. coli DnaK-mutantstammer tjener som ideelle modeller til undersøgelse af Hsp70's chaperonaktivitet gennem en komplementationstilgang. Da DnaK kun er afgørende under stressende forhold, udføres komplementationsanalysen typisk ved forhøjede temperaturer (figur 1). Nogle fordele ved at bruge E. coli til denne undersøgelse omfatter dets velkarakteriserede genom, hurtig vækst og de lave omkostninger ved dyrkning og vedligeholdelse17.
I denne artikel beskriver vi detaljeret en protokol, der involverer brugen af E. coli dnaK756-celler til at studere funktionen af Hsp70. De HSP70'ere, vi anvendte i analysen, er vildtype DnaK og dets kimære derivat, KPf (bestående af ATPase-domænet for DnaK smeltet sammen med det C-terminale substratbindende domæne af Plasmodium falciparum Hsp70-1 6,18). KPf-V436F blev heterologt udtrykt som en negativ kontrol, da mutationen i det væsentlige blokerer den fra bindende substrater og dermed ophæver dens chaperonaktivitet9.

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			<td>HG000BX4.250</td>
			<td>Constituent of medium and liquid growth assay</td>
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			<td>Merck</td>
			<td>SAAR5042020EM</td>
			<td>Constituent of PBS buffer</td>
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			<td>Thermofischer scientific</td>
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			<td>SAAR5822320EM</td>
			<td>Constituent of medium and liquid growth assay</td>
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			<td>Merck</td>
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escherichia coli -based complementation assay to study the chaperone function of heat shock protein 70

Varmechokprotein 70 (Hsp70) er et konserveret protein, der letter foldningen af andre proteiner i cellen, hvilket gør det til en molekylær chaperon. Mens Hsp70 ikke er afgørende for E. coli-celler , der vokser under normale forhold, bliver denne chaperon uundværlig for vækst ved forhøjede temperaturer. Da Hsp70 er meget bevaret, er en måde at studere chaperonfunktionen af Hsp70-gener fra forskellige arter på heterologt at udtrykke dem i E. coli-stammer , der enten mangler Hsp70 eller udtrykker en indfødt Hsp70, der er funktionelt kompromitteret. E. coli dnaK756 celler er ude af stand til at understøtte λ bakteriofag DNA. Desuden udviser deres oprindelige Hsp70 (DnaK) forhøjet ATPase-aktivitet, samtidig med at de viser reduceret affinitet for GrpE (Hsp70-nukleotidbytterfaktor). Som følge heraf vokser E. coli dnaK756-celler tilstrækkeligt ved temperaturer fra 30 ° C til 37 ° C, men de dør ved forhøjede temperaturer (>40 ° C). Af denne grund tjener disse celler som en model til undersøgelse af Hsp70's chaperonaktivitet. Her beskriver vi en detaljeret protokol for anvendelsen af disse celler til at gennemføre et komplementationsassay, der muliggør undersøgelsen af in cellulo chaperonfunktionen af Hsp70.

Heat shock proteins play an important role as molecular chaperones by facilitating protein folding, preventing protein aggregation, and reversing protein misfolding1,2. Heat shock protein 70 (Hsp70) is one of the most prominent molecular chaperones, playing a central role in protein homeostasis3,4. DnaK is the E. coli Hsp70 homologue5.
Various biophysical, biochemical, and cell-based assays have been developed to explore the chaperone activity of Hsp70 and to screen for inhibitors targeting this chaperone6,7,8. Hsp70 is a highly conserved protein. For this reason, several Hsp70s of eukaryotic organisms, such as Plasmodium falciparum (the main agent of malaria), have been reported to substitute for DnaK function in E. coli6,9. In this way, an E. coli-based complementation assay has been developed involving the heterologous expression of Hsp70s in E. coli to explore their cytoprotective function. Typically, this assay involves the utilization of E. coli cells that are either deficient for DnaK or that express a native DnaK that is functionally compromised. While DnaK is not essential for E. coli growth under normal conditions, it becomes essential when the cells are grown under stressful conditions such as elevated temperatures or other forms of stress10,11.
E. coli strains that have been developed to study Hsp70 function using a complementation assay include E. coli dnaK103&#160;(BB2393 [C600 dnaK103(Am) thr::Tn10]) and E. coli dnaK756. E. coli dnaK103&#160;cells produce a truncated DnaK that is non-functional, and as such, the cells grow adequately at 30 &#176;C, while the strain is sensitive to cold and heat stress12,13. Similarly, the E. coli dnaK756/BB2362 (dnaK756&#160;recA::TcR Pdm1,1) strain does not grow above 40 &#176;C14,15. The E. coli dnaK756&#160;strain expresses a mutant native DnaK (DnaK756) characterized by three glycine-to-aspartate substitutions at positions 32, 455, and 468, giving rise to compromised proteostatic outcomes. Consequently, this strain is resistant to bacteriophage &#955; DNA14. Additionally, E. coli&#160;dnaK756 exhibits elevated ATPase activity, while its affinity for the nucleotide exchange factor, GrpE, is reduced16. E. coli DnaK mutant strains serve as ideal models for investigating the chaperone activity of Hsp70 through a complementation approach. Since DnaK is only essential under stressful conditions, the complementation assay is typically conducted at elevated temperatures (Figure 1). Some advantages of using E. coli for this study include its well-characterized genome, rapid growth, and the low cost of culturing and maintenance17.
In this article, we describe in detail a protocol involving the use of E. coli dnaK756&#160;cells to study the function of Hsp70. The Hsp70s we employed in the assay are wild-type DnaK and its chimeric derivative, KPf (made up of the ATPase domain of DnaK fused to the C-terminal substrate-binding domain of Plasmodium falciparum Hsp70-16,18). KPf-V436F was heterologously expressed as a negative control since the mutation essentially blocks it from binding substrates, thus abrogating its chaperone activity9.

Forfatter Spotlight: Udforskning af varmechokproteiner i malaria- og tuberkuloseinfektioner

Escherichia coli-baseret komplementationsanalyse til undersøgelse af chaperonfunktionen af varmechokprotein 70

I study the role of heat shock proteins in conferring future static protection to antigens that cause human infections such as malaria and tuberculosis. My research essentially focuses on the structure and function features of the heat shock protein machinery of these pathogens that cause human infections. Heatstroke proteins are generally highly concept, however, they display some level of functional specialization.In addition, they are implicated in drug resistant. As such, there are ongoing efforts to target them towards reversing drug resistance in various disease models. They include biochemical, biophysical, bioinformatics, and cell biologic techniques.In other words, various areas of techniques are used to study the roles of these proteins. In our case, the study of proteins of Plasmodium was the main agent of malaria and that they are difficult to produce and this limits the capability to study these proteins. For example, it is difficult to generate sufficient levels to crystallize and image the proteins.We have established some of the functional networks of these proteins in the malaria parasite. We have also established assays to study the compounds targeting these proteins in agents causing malaria and TB.The current protocol is used to explore the chaperone protein folding role and then cytoprotective function of HSP 70 using equal as a model. The protocol could also be adopted for screening small molecule inhibitors targeting protein 70.

Figur 2 viser et billede af de scannede agarholdige celler, der blev spottet og dyrket ved den tilladelige væksttemperatur på henholdsvis 37 °C og 43,5 °C. På højre side af figur 2 repræsenterer udskårne vestlige blotkomponenter ekspressionen af DnaK-, KPf- og KPf-V436F i E. coli dnaK756-celler. Som forventet lykkedes det alle E. coli dnaK756-celler, der blev dyrket ved den tilladelige væksttemperatur på 37 °C, at vokse. Under de ikk...

Watch this Scientific Journal Video about Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections at JoVE.com

Denne protokol demonstrerer chaperonaktiviteten af varmechokprotein 70 (Hsp70). E. coli dnaK756-celler tjener som model for analysen, da de har en indfødt, funktionelt nedsat Hsp70, hvilket gør dem modtagelige for varmestress. Den heterologe introduktion af funktionel Hsp70 redder cellernes vækstmangel.

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. ...

Jeg studerer varmechokproteiners rolle i at give fremtidig statisk beskyttelse til antigener, der forårsager humane infektioner som malaria og tuberkulose. Min forskning fokuserer hovedsageligt på strukturen og funktionsfunktionerne i varmechokproteinmaskineriet i disse patogener, der forårsager infektioner hos mennesker. Hedeslagproteiner er generelt meget koncept, men de viser et vist niveau af funktionel specialisering.Derudover er de impliceret i lægemiddelresistente. Som sådan er der igangværende bestræbelser på at målrette dem mod at vende lægemiddelresistens i forskellige sygdomsmodeller. De omfatter biokemiske, biofysiske, bioinformatik og cellebiologiske teknikker.Med andre ord bruges forskellige områder af teknikker til at studere disse proteiners roller. I vores tilfælde var undersøgelsen af proteiner fra Plasmodium det vigtigste middel til malaria, og at de er vanskelige at producere, og dette begrænser evnen til at studere disse proteiner. For eksempel er det svært at generere tilstrækkelige niveauer til at krystallisere og afbilde proteinerne.Vi har etableret nogle af de funktionelle netværk af disse proteiner i malariaparasitten. Vi har også etableret assays for at studere forbindelserne rettet mod disse proteiner i stoffer, der forårsager malaria og TB. Den nuværende protokol bruges til at udforske chaperonproteinets folderolle og derefter cytoprotektive funktion af HSP 70 ved hjælp af lige som model. Protokollen kunne også vedtages til screening af små molekylehæmmere rettet mod protein 70.

escherichia-coli-based-complementation-assay-to-study-chaperone

Research

JoVE Journal

Biochemistry

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70&#160;genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756&#160;cells are unable to support &#955; bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756&#160;cells grow adequately at temperatures ranging from 30 &#176;C to 37 &#176;C, but they die at elevated temperatures (&#62;40 &#176;C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.